The present invention is directed to the preparation of a class of membranes or filters to which enzymes and other molecules of biological activity are attached by chemical bonds, wherein the process of activation of the membrane pore surfaces (where such is needed) and/or the process of coupling are carried out under the imposition of a pressure gradient across the membrane, and where the utilization of this system can be effected similarly by forcing the substrate to be treated through the membrane pores under pressure. These enzyme-coupled ultrafiltration (hereafter ECUF) membranes are applicable, in principle, to all of those processes wherein enzymes are used, especially when combined with processes of ultrafiltration.
Conventional uses of enzymes employ them in microbial and other cells or as impure or pure soluble enzyme preparations. In recent years enzymes have been entrapped in insoluble gels or coupled chemically to either soluble polyelectrolytes or to insoluble surfaces or within gels of cellulose, glass and other substances. Further, whole cells have been similarly stabilized. It has been found that enzymes stabilized in this manner often show altered characteristics of a favorable nature, in that their effective pH ranges are altered or broadened, their chemical stability is improved as regards chemical (as to urea) and thermal degradation, and thus they can in principle be used for longer periods of time as contrasted to the usages of conventional fermentation processes. All of these advantages are well known and are summarized in various articles and books, especially "Immobilized Enzymes" by O. R. Zaborsky, CRC Press, Cleveland, Ohio 1974.
All of these enzymatic systems and processes may be characterized by the Figure of Merit (FOM) of an enzyme reactor, namely the weight or volume fraction of the reactor which is enzyme, times the ratio of the activity of the same native enzyme in solution at comparable pH and temperature levels. The FOM is proportional to the amount of moles of substrate converted per unit time for given weight or volume of reactor.
The usual process employed heretofore has been to couple enzymes to spherical gel particles of agarose, dextran or of porous glass. These particles are usually quite small, about 20 to 100 microns in diameter to minimize the rate of diffusion as much as is practical. The gel particles of most polymers are quite soft because they are largely water, up to about 95% water in the uncoupled state. Gel particles of glass and other inorganic materials are not soft, but they do not usually contain as large a void fraction.
The usual means of coupling involves reacting the gel bead with an activating chemical or chemicals, removing excess activating chemicals by rinsing the beads for an appropriate period of time, soaking the beads in the enzyme solution whereby the enzyme diffuses into the beads and coupling takes place, following which the unreacted enzyme is flushed from the beads. In use the beads are slurried with the substrate solution or, more frequently, they are placed in a column and the substrate is passed through it. The fineness of the beads produces a low hydraulic permeability for the column and, if one uses a high pressure to increase the rate of throughput, distortion and/or a partial destruction of the beads can result. One can employ filters to separate beads from solution or make the beads dense so a fluidized bed can be operated, but this requires more and expensive equipment and/or a lower loading or % weight or volume % of the system which is enzyme.
While one can coat the outer external surfaces of particles, fibers or films to render the enzyme there readily accessible to the substrate from the solution and thus reduce the time required for diffusion, the loading with enzyme is very low because a thin monomolecular coat of enzyme, even on a rough surface, does not involve a large amount of enzyme.
Further, when enzymes are coupled into gels or entrapped in them by physical means the extent of coupling is not uniform because of the intrinsically random nature of the process so some enzyme molecules are undoubtedly coupled or entrapped less than are others and a leaching or loss of enzyme from the column or bead slurry is usually encountered.
For these reasons, the FOM reported frequently is that corresponding to 3% loading and 3% activity or FOM= 9.times.10.sup..sup.-4 . While some report higher loadings there is usually a loss in activity so a FOM of 10.sup..sup.-2 is considered high. It must be emphasized that the FOM is an operational parameter; it describes the overall activity or rate of conversion of a reactor under conditions of use, not the activity which could be attained if diffusion were not rate-controlling. Indeed, in all but the surface-coated systems, diffusion is almost invariably rate-determining, both in the activation-coupling processes and in processes of use.
It is accordingly an object of the present invention to provide coupled enzyme systems which are useful for a variety of applications, i.e. virtually all of the applications to which stablized enzymes have heretofore been put.